Many lysosomal storage disease-associated mutant enzymes exhibit compromised endoplasmic reticulum (ER) folding and therefore are subjected to degradation instead of being trafficked to the lysosome, where they normally degrade their substrates. This leads to lysosomal substrate accumulation and thus, pathology. In Specific Aim 1 we aspire to identify proteostasis network components responsible for (1) folding wild type and Gaucher disease-associated mutant ?-glucocerebrosidases (GCs) in the ER, (2) trafficking them through the Golgi and on to the lysosome, (3) stabilizing/activating them in the lysosome, and (4) degrading misfolded GC in the ER by immunoisolating the GC interactome (antibody recognizes the extreme C-terminus and is thus conformation and mutant insensitive) and identifying the proteins by mass spectrometry. Mutant ?- glucocerebrosidases exhibit overlapping interactomes with distinct members because they accumulate in various subcellular compartments. GC interacting partners, prioritized on the basis of 4 methods, will be RNAi depleted in L444P GC cells to discern what extent GC proteostasis is perturbed using the intact cell GC activity assay, endo H sensitivity and immunofluorescence colocalization. Several novel GC proteostasis network components have already been identified, allowing several hypotheses regarding the GC proteostasis network to be tested. Simply lowering the growth temperature of patient-derived cells to 300C, inducing the unfolded protein response (UPR), or increasing ER Ca2+ concentration with ryanodine antagonists enhances mutant enzyme folding, trafficking and function enabling the interactome of adapted cells vs. controls to be determined to test numerous hypotheses about mechanism, including whether these results extend to other lysosomal storage diseases. In Specific Aim 2, we explore which genetically-encodable, small molecule regulated UPR- associated transcription factors individually and in combination can enhance mutant lysosomal enzyme folding, trafficking and function. Understanding the transcriptome upregulated by these transcription factors and combinations affording heterodimeric transcription factors cross referenced to the upregulated proteome in proteostasis network adapted cells will provide additional mechanistic insight into the enhancement in proteostasis. Unregulated overexpression of XBP1s restores L444P GC proteostasis motivating the development of aryl oxime ether IRE1 activators that will be tested in patient-derived cells from multiple lysosomal storage diseases to discern efficacy. IRE1 Inhibitors should be useful to cancer researchers. PUBLIC HEALTH RELEVANCE: This research defines the protein homeostasis network that can be altered to enhance mutant lysosomal storage disease-associated enzyme folding, trafficking and function. Adapting the protein homeostasis network with small molecules offers the possibility of ameliorating multiple lysosomal storage diseases of similar etiology by fixing the folding and trafficking of the mutant enzyme instead of replacing the enzyme-the current standard of care, which is expensive, disease specific and not useful for alleviating neuropathic lysosomal storage diseases. Small molecule adaptors of cellular protein homeostasis used in combination with pharmacologic chaperones that bind to and stabilize a particular protein are envisioned to be a very promising future approach for treating loss-of-function diseases, of which the lysosomal storage diseases of focus herein are one class.